Secretory diarrheas are frequently the consequence of inappropriate intracellular second messenger signaling (cAMP and/or Ca2+) in intestinal and colonic epithelial cells. These signals are thought to activate apical Ca2+- and cAMP-dependent anion-conducting pathways (e.g. Cl- channels such as CFTR) that drive fluid secretion into the gut lumen, resulting in potentially life-threatening loss of water and nutrients. Many of the diverse agents (intestinal pathogens, chemotherapy drugs, bioactive dietary constituents, bile acids, inflammatory mediators and environmental toxins) that cause diarrhea also cause the release of Ca2+ from the endoplasmic reticulum (ER). We recently described a novel signaling pathway that connects the free concentration of Ca2+ in the lumen of the ER to the activation of the enzyme that generates cAMP, adenylyl cyclase (AC). This process is completely independent of cytosolic Ca2+, and requires the translocation of an ER Ca2+ sensor protein, STIM1. Our central hypothesis is that persistent Ca2+ store depletion contributes to the abnormal anion secretion associated with diarrheal diseases not through elevation in cytosolic Ca2+, but rather through "store- operated" cAMP signaling. In order to evaluate this possibility, we will further characterize this novel pathway by identifying its individual protein components and their spatial relationships with one another at the subcellular level. The specific aims of this project are: (i) To identify the specific AC isoform(s) involved in this process, and to pinpoint their localization and their spatial relationships with respect to the ER and the STIM proteins;this will include measurements of putative micro-domains of cAMP production at the subcellular level using TIRF and targeted FRET-based cAMP sensors;(ii) To evaluate the participation of other binding partners of STIM1 (such as the Orai family of Ca2+ channels) in this process;and (iii) Using novel tools generated in our lab for controlling cAMP levels, to evaluate the extent to which the cAMP produced following store depletion drives Cl- secretion in colonic epithelial cells. Public Health Relevance: Diarrhea is the second leading cause of death in infants worldwide and is a significant source of morbidity and mortality in the U.S., particularly in vulnerable patient populations. We are seeking exploratory/development funding for this project because the concept of a store-operated cyclic nucleotide pathway is a dramatic departure from the conventionally accepted doctrine used to explain the genesis of secretory diarrheas. These paradigm-shifting studies may ultimately lead to new avenues for treatment and prevention of diarrhea and other diseases. PUBLIC HEALTH RELEVANCE: Diarrhea is the second leading cause of death in infants worldwide and is a significant source of morbidity and mortality in the U.S., particularly in vulnerable patient populations. We are seeking exploratory/development funding for this project because the concept of a store- operated cyclic nucleotide pathway is a dramatic departure from the conventionally accepted doctrine used to explain the genesis of secretory diarrheas. These paradigm-shifting studies may ultimately lead to new avenues for treatment and prevention of diarrhea and other diseases. )